Linear chromosomes terminate in specialized nucleoprotein structures called telomeres. These natural ends are integral to genomic stability and cellular proliferation, and their dysregulation is linked to cancer, aging and other human diseases. Telomeric DNA is unusually GT-rich, and ends in a 32 single-strand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. Furthermore, telomeres cannot be fully replicated by the canonical DNA replication machinery, leading to erosion of telomere sequence with every cell cycle. Highly proliferating cells overcome this limitation through the action of the replicative enzyme telomerase. This research program targets two outstanding questions of telomere maintenance. First, how do the components of the telomerase holoenzyme contribute to telomerase action? Second, how do the telomere capping factors shield the end of the chromosome from detection as compromised DNA? Biochemical, structural and genetic strategies are combined to understand how telomere factors perform these activities. This integrated program is performed a model system amenable to multiple levels of characterization. The first Aim investigates the specific biochemical and structural roles of two proteins required for telomerase action in vivo that are not directly responsible for catalysis. We will test the hypothesis that the primary role of one factor of the holoenzyme is to bring telomerase to its site of action through both protein/protein and protein/nucleic acid interactions. The second Aim surrounds the function of telomere capping factors. Our structural studies strongly suggest that these capping factors adopt tertiary and quartenary structures remarkably similar to those of replication protein A (RPA), despite no discernible sequence similarities. Using the RPA model as a motivating hypothesis, the putative capping complex will be evaluated for its biochemical ability to functionally replace RPA, as well as for the presence of telomere- specific features. In the final Aim, knowledge derived from the structural and biochemical studies is applied in a highly controllable reconstituted telomerase assay to understand the origins of telomerase activity and processivity. This line of research will assess the interplay between processes that promote telomerase activity and those acting to suppress it. Throughout the research program, insights derived from these studies are validated in vivo using genetic tools and analyses of a variety of telomere phenotypes. This hypothesis-driven, highly unified research program will provide novel insights into the fundamental processes that maintain a central mechanism of chromosome stability. PUBLIC HEALTH RELEVANCE: Telomeres, the ends of linear chromosomes, play key roles in cancer and aging due to their ability to discriminate natural DNA ends from damaged DNA and by compensating for the inability of the standard replication machinery to fully copy the chromosomal terminus. The length of the telomere is a marker for aging, while the ability to properly cap telomeres is associated with genomic stability. The vast majority of human cancers activate the replicative enzyme telomerase to overcome the natural brake telomeres place on cellular proliferation. As a result of these activities, proper telomere function is integral to huan health.